Immune subset-committed proliferating cells populate the human foetal intestine throughout the second trimester of gestation

The intestine represents the largest immune compartment in the human body, yet its development and organisation during human foetal development is largely unknown. Here we show the immune subset composition of this organ during development, by longitudinal spectral flow cytometry analysis of human foetal intestinal samples between 14 and 22 weeks of gestation. At 14 weeks, the foetal intestine is mainly populated by myeloid cells and three distinct CD3–CD7+ ILC, followed by rapid appearance of adaptive CD4+, CD8+ T and B cell subsets. Imaging mass cytometry identifies lymphoid follicles from week 16 onwards in a villus-like structure covered by epithelium and confirms the presence of Ki-67+ cells in situ within all CD3–CD7+ ILC, T, B and myeloid cell subsets. Foetal intestinal lymphoid subsets are capable of spontaneous proliferation in vitro. IL-7 mRNA is detected within both the lamina propria and the epithelium and IL-7 enhances proliferation of several subsets in vitro. Overall, these observations demonstrate the presence of immune subset-committed cells capable of local proliferation in the developing human foetal intestine, likely contributing to the development and growth of organized immune structures throughout most of the 2nd trimester, which might influence microbial colonization upon birth.


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Policy information about availability of computer code Data collection Data for flow cytometry were acquired using an a 5-laser Cytek® Aurora cytometor (BD Biosciences). Data for imaging mass cytometry were acquired using a Hyperion imaging-mass cytometer (Fluidigm) at 1 μm resolution following the manufacturer's instructions.
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Reporting on sex and gender
There are no information on sex and gender of the fetal samples, which are anonymous.

Population characteristics
Fetal intestinal tissues were obtained from elective abortions with informed consent. The gestational age ranged from 14 to 22 weeks. The gender of fetus is unknown.

Recruitment
The patients who did elective abortions in the Contraception, Abortion and Sexuality (CASA) in Leiden and The Hague were included after informed consent. The gestational age of the fetuses ranged from 14 to 22 weeks.

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The work described here was reviewed and approved by the Medical Ethical Committee of Leiden University Medical Centre (P08.087). All experiments were conducted in accordance with local ethical guidelines and the principles of the Declaration of Helsinki.
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Sample size
In this study no statistical methods were used to predetermine sample size and a total of ~50 unique human fetal intestinal samples were used. For the high-dimensional single-cell assays the number was chosen to obtain sufficient number of immune cells to capture the heterogeneity. For the cell proliferation assays and functional profile studies, replicates were included to reproduce findings in independent experiments. All replication attempts were successful. See Figure legends for these specifications. In addition, 8 fetal intestinal samples were used for imaging mass cytometry and RNAscope analysis to investigate the immune composition and lymphoid follicles. 28 fetal, 2 pediatric and 6 adult samples were analyzed by single cell spectral flow cytometry to compare immune composition.
Data exclusions In accordance with current practice in the field, single cells which failed standard QC for flow cytometry in OMIQ were excluded from analysis.

Replication
All attempts at replication were successful. And at least 3 independent experiments were performed for every finding.
Randomization All experiments were performed using randomly selected human fetal intestinal samples with different gestational weeks, non-inflamed intestinal samples from pediatric and adult patients.

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No blinding test in this study. Since all the samples were selected and grouped randomly and data collection and /or analysis were mainly performed using computational tool, we consider that blinding was not applicable to the current study.

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We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation All antibodies used in flow cytometry were verified by staining human peripheral blood mononuclear cell and/or tonsil cells with known expression or not. Self-conjugation antibodies with metal used in imaging mass cytometry were validated on frozen intestinal tissue by IHC before conjugation, and pre-conjugation antibodies were purchased from Fluidigm.

Flow Cytometry
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March 2021
Methodology Sample preparation All the immune cells were isolated from human fetal intestines and stored in liquid nitrogen. Before staining, the cells were thawed and stained with specific panel as described in Methods: The 26-antibody flow cytometry-based panel was developed for in-depth immunophenotyping of the major cell subsets present in the human fetal intestine through time. In total, 3 experiments were performed for immunophenotypic studies of 28 human fetal intestinal samples. Antibodies used for spectral flow cytometry with a 5-laser Cytek® Aurora are listed in Supplementary Table 1. For surface staining, single-cell suspensions of fetal intestinal samples were incubated with fluorochrome-conjugated antibodies and human Fc block (BioLegend) for 30 min at 4 °C. After washing, cells of samples were then fixed/permeabilized using Foxp3 Staining Buffer Set, according to manufacturer's instructions (ThermoFisher). For intracellular staining, the fixed/permeabilized cells were incubated with the antibodies for 45 min at 4 °C, followed by washing of the cells with permeabilization buffer. Then the stained cells were resuspended. Reference samples were incorporated and individually stained by UltraComp eBeadsTM Compensation Beads (ThermoFisher), PBMCs or cells from human tonsils. After the completion of the sample preparation, the samples were immediately acquired using a 5-laser Cytek® Aurora (BD Biosciences). Data were analyzed to check quality with FlowJo software version 10.6 (Tree Star Inc). We utilized OMIQ to perform the high-dimensional analysis for human fetal intestinal samples (https://www.omiq.ai/).

Gating strategy
All acquired cells were first gated on FCS/SSC and FCS/FCH, then L/D dye to obtain the single live cells. Every Flow cytometry experiment, a peripheral blood mononuclear cell control sample was included as staining and gating control. Based on the unstained sample and controls to set the positive and negative gates.
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